The rapid acquisition and analysis of high volumes of data in biological samples had its advent in the early days of the human genome sequencing project. Microarray technology has facilitated the interrogation of large numbers of samples for biologically relevant patterns in a variety of physiological, drug-induced or clinically relevant cellular states. A challenge has now presented itself with respect to how these large volumes of information can be integrated into an accurate model of cellular behavior and processes. For example, information relating the effect of a drug to the extent and duration of apoptosis in cancer cells would be invaluable information in a screen for cancer drugs. Similarly, information of cytoskeletal changes leading to invasiveness would greatly streamline the development of an efficient anti-angiogenic drug strategy.
The discipline of cytomics has emerged to meet these and other demands in both the academic and industrial research communities. The importance of cytomics derives from the fact that the cell is the minimal functional unit within our physiology. An attendant technology to the emergence of cytomics is High Content Screening (HCS) which is generally defined as a simultaneous, or near real-time, multiparametric analysis of various aspects of cell state.
The complexity of cell function is only part of why cytomics will likely become a major field of study in the near future. Every cell is different, and by studying each cell's unique function, that cell type can be further modeled for subsequent analysis using statistical techniques. Within a short time, the inventors herein forecast that most pharmaceutical companies will not operate without encompassing the essential features of cytomics-drugs-design; a process that will increasingly operate at the level of modified cellular functions. Future cancer strategies may place greater emphasis on cytome-alignment or cytomic-realignment, which may be viewed as the “cellular form” of tissue engineering. Such an approach will require a better-than-ever understanding of how the cell operates, of how to measure cell function, and of how to characterize a live cell in minute detail. To meet this challenge, there is need in the art for the development of new technologies and new analytical tools for exquisitely sensitive single-cell analysis.
A primary goal of cytomics is the discovery of functional relationships between the cell (cytome) and the metabolic pathways (i.e., proteomics, which enables rapid identification of proteins from specific cell populations) resulting from genetic control mechanisms (i.e., genomics; some in the art relate cytomics to functional genomics). With cytomics, the amount of information being collected from the cell is expanded in order to obtain functional data, not just morphological, phenotypic, or genotypic data.
Currently, there are two major branches of cytomics: analytical cytology and image cytology. The first, analytical cytology, is comprised of traditional analytical techniques such as: flow cytometry, single cell analysis systems and tissue analysis (after cell separation). The second, image cytology (and analysis) is comprised of techniques such as “quantitative” fluorescence assays, high throughput cell culture assays (96-384-1536 well plates), drug effect assays of cytotoxicity, toxicology assays, apoptosis assays, cell proliferation assays, cell ploidy assays, and DNA array assays. These techniques are typically applied to single cells, tissues and sections, and cell culture systems in both 3D and 4D cell culture environments. Laser Scanning Cytometry (LSC) is a well-known example of this type of assay.
At the highest level, cytomics links technology to functional biology at the cellular level by relating measurement and detection to structure and function. To achieve this end, cytomics integrates tools like flow cytometry, image cytometry, etc. with proteomics and this brings together traditional cytometry and non-traditional cytometry. With the application of so many different measurement technologies to the same problem, informatics now assumes a primary rather than a secondary role in cytomics. For instance, in a typical flow cytometry system, there are 120,000 events per second per output channel, with measurements being acquired for multiple channels. Another example is offered by very high speed cell culture plate imaging systems applied to detect fluorescent markers in cells.
The term HCS is used to differentiate assays that use live cells and to provide single point readouts (e.g., High Throughput Screening (HTS) assays), which are often based on the biochemistry of ligand binding. HCS combines cell-based arrays with robotics, informatics, and advanced imaging to provide richly detailed information on cell morphology and other responses in large quantities.
Many protocols for generating data are already well developed in their respective disciplines, from quantitative Polymerase Chain Reaction (PCR), to flow cytometry, to antibody staining. The methods for acquisition of this data, such as different types of optical microscopy, have already undergone extensive development. Perhaps the most important image acquisition methods for HCS relate to cellular imaging, including drug effect assays for cytotoxicity, apoptosis, cell proliferation, and nucleocytoplasmic transport. Frequently, these approaches utilize cell sensors based on fluorescent proteins and dyes, and thus provide researchers with an ability to screen drugs and to answer more complex biological questions such as target identification and validation and to investigate gene and protein function.
In an effort to fill a need in the art for improved cellular imaging techniques, the inventors herein disclose a new, inexpensive, and easy-to-use imaging technology suitable for simultaneous capture of multiple measurements from individual cells that will enable molecular colocalization, metabolic state and motility assessment, and determination of cell cycle, texture, and morphology. This technology will be capable of not only HCS, but also permit selection of single cells for subsequent high-resolution imaging based on the outputs of the HCS. By increasing the analytical resolution to assess the sub-cellular state in vivo, the inventors herein hope to increase biological resolution by providing a means to follow the location, timing, and interdependence of biological events within cells in a culture.
The present invention builds upon the previous works by one of the inventors herein, wherein the extraordinary magnetoresistance (EMR) and extraordinary piezoconductance (EPC) properties of hybrid semiconductor/metal devices were used to develop improved sensing techniques for a wide variety of applications. For EMR devices, examples include but are not limited to read heads for ultra high density magnetic recording, position and rotation sensors for machine tools, aircraft and automobiles, flip phone switches, elevator control switches, helical launchers for projectiles and spacecraft, and the like. For EPC devices, examples includes but are not limited to a myriad of pressure sensors, blood pressure monitors, and the like. See U.S. patent application publication 2004/0129087 A1 entitled “Extraordinary Piezoconductance in Inhomogeneous Semiconductors”, U.S. Pat. Nos. 6,714,374, 6,707,122, 5,965,283, and 5,699,215, Solin et al., Enhanced room-temperature geometric magnetoresistance in inhomogeneous narrow-gap semiconductors, Science, 2000; 289, pp. 1530-32; Solin et al., Self-biasing nonmagnetic giant magnetoresistance sensor, Applied Physics Letters, 1996; 69, p. 4105-4107; Solin et al., Geometry driven interfacial effects in nanoscopic and macroscopic semiconductor metal hybrid structures: Extraordinary magnetoresistance and extraordinary piezoconductance, Proc. of the International Symposium on Clusters and Nanoassemblies, Richmond, 2003; Rowe et al., Enhanced room-temperature piezoconductance of metal-semiconductor hybrid structures, Applied Physics Letters, 2003; 83, pp. 1160-62; Solin et al., Non-magnetic semiconductors as read-head sensors for ultra-high-density magnetic recording, Applied Physics Letters, 2002; 80, pp. 4012-14; Zhou et al., Extraordinary magnetoresistance in externally shunted van der Pauw plates, Applied Physics Letters, 2001; 78, p. 667-69; Moussa et al., Finite element modeling of enhanced magnetoresistance in thin film semiconductors with metallic inclusions, Physical Review B (Condensed Matter and Materials Physics) 2001; 64, pp. 184410/1-184410/8; Solin et al., Room temperature extraordinary magnetoresistance of non-magnetic narrow-gap semiconductor/metal composites: Application to read-head sensors for ultra high density magnetic recording, IEEE Transactions on Magnetics, 2002; 38, pp. 89-94; Pashkin et al., Room-temperature Al single-electron transistor made by electron-beam lithography, Applied Physics Letters, 2000; 76, p. 2256-58; Branford et al., Geometric manipulation of the high field linear magnetoresistance in InSb epilayers on GaAs (001), Applied Physics Letters, 2005, 86, p. 202116/1-202116/3; and Rowe et al, A uni-axial tensile stress apparatus for temperature-dependent magneto-transport and optical studies of epitaxial layers, Review of Scientific Instruments, 2002; 73, pp. 4270-76, the entire disclosures of each of which being incorporated by reference herein.
The inventors herein extend upon the EMR and EPC sensors referenced above to disclose arrays comprised of a plurality of individual hybrid semiconductor/metal devices that can be used to measure voltage responses that are indicative of various characteristics of an object that is in proximity to the hybrid semiconductor/metal devices (such as one or more cells, either in vivo or in vitro) and from which images of the object characteristics can be generated. These hybrid semiconductor/metal devices may comprise a plurality of EXX sensors on a microscale or a nanoscale. Preferably, these EXX sensors comprise nanoscale EXX sensors. As used herein, “nanoscale” refers to dimensions of length, width (or diameter), and thickness for the semiconductor and metal portions of the EXX sensor that are not greater than approximately 1000 nanometers in at least one dimension. As used herein, “microscale” refers to dimensions of length, width (or diameter), and thickness for the semiconductor and metal portions of the EXX sensor that are not greater than approximately 1000 micrometers in at least one dimension. The term “EXX sensor” refers to a class of hybrid semiconductor/metal devices having a semiconductor/metal interface whose response to a specific type of perturbation produces an extraordinary interfacial effect XX or an extraordinary bulk effect XX. The interfacial or bulk effect XX is said to be “extraordinary” as that would term would be understood in the art to mean a many-fold increase in sensitivity relative to that achieved with a macroscopic device for the same perturbation. Examples of XX interfacial effects include the MR (magnetoresistance) and PC (piezoconductance) effects known from previous work by one of the inventors herein as well as EC (electroconductance) effects. It should be noted that AC (acoustoconductance) effects are effectively the same as the PC effects in that both the EAC and EPC devices can have identical structure. An EAC device can be thought of as a subset of a class of EPC devices, wherein the EAC device is designed to respond to a strain perturbation that is produced by an acoustic wave. An example of an XX bulk effect includes OC (optoconductance) effects. Thus, examples of suitable nanoscale EXX sensors for use in the practice of the present invention include nanoscale EMR sensors, nanoscale EPC sensors, nanoscale EAC sensors, nanoscale EOC sensors, and nanoscale EEC sensors.
The inventors herein believe that the use of nanoscale EAC sensors and nanoscale EPC sensors in an imaging array will provide improved imaging resolution, improved signal-to-noise ratio (SNR), and higher bandwidth than conventional ultrasonic or other modes of detectors. Accordingly, the use of an array having a plurality of nanoscale EAC sensors and/or a plurality of nanoscale EPC sensors can be used for a myriad of applications, including but not limited to in vitro cell imaging, in vivo invasive catheter-based applications for medical imaging, endoscopic imaging for gastrointestinal, prostate, or urethral/bladder/ureteral applications, transdermal medical imaging for disease characterization, detection of abnormal cells in serum samples, acoustic imaging, pressure sensing in nanofluidics, and blood pressure monitoring inside small vessels.
The inventors herein further believe that the use of nanoscale EOC sensors in an imaging array will produce ultra high resolution images of individual cells or tissues that are indicative of the presence of fluorescence in the cells/tissues, a result that can be highly useful in the investigation of cancer and cancer therapeutics, optical microsccopy, photosensors and photodetectors, image intensifiers, position sensitive detectors, and position and speed control systems. The inventors further believe that additional uses for nanoscale EOC sensors in an imaging array include their use in static charge detection, EM radiation sensors, and EKG sensors.
The inventors herein further believe that the use of nanoscale EEC sensors in an imaging array will produce ultra high resolution images of electric charge distribution over the surface of one or more living cells, a result that can provide valuable information for monitoring cancer metastasis and targeted drug delivery, particularly so when a series of such images are taken over time to track the progression of the cell's electric charge over time. The inventors herein believe that the nanoscale EEC sensors of the present invention will serve as a significantly more accurate and effective measure of cell electric charge than the conventional electrophoresis technique that is known in the art because electrophoretic measurements suffer from a complicated instrumental dependence and a lack of spatial resolution.
The inventors herein further believe that the use of nanoscale EMR sensors in an imaging array will produce ultra high resolution images of magnetoresistance over the surface of one or more living cells, a result that can provide valuable information for studying the magnetic fields produced by nonmagnetic particles embedded in cancer cells, for monitoring magnetically labeled nanoparticles that are trafficking inside the cells or for sensing the evolution of imposed magnetic resonance spin orientations.
As perhaps the most powerful embodiment of the present invention, the inventors herein envision that a multi-modal array having a plurality of different types of EXX sensors can be used to simultaneously (or nearly simultaneously) generate multiple images that are representative of different characteristics of one or more cells that are imaged by the array. For example, with a multi-modal array having a plurality of EOC sensors and a plurality of EEC sensors, multiple images can be simultaneously generated that are representative of both fluorescent emissions by the cell(s) and the surface charge of the cell(s). Such images would exhibit a nanoscale resolution. As used herein, the term “type” as used in connection with EXX sensors refers to the type of XX interfacial effect or bulk effect relied upon by the sensor. For example, an EAC sensor is of a different type than an EEC sensor.
The inventors further note that the ultra high resolution images produced in the practice of the present invention can not only be two-dimensional images, but optionally can also be three-dimensional images through the use of confocal imaging techniques.
These and other features and advantages of the present invention will be described hereinafter to those having ordinary skill in the art.